As part of a continuing study of the neurophysin-peptide hormone system, the tripeptide analogue of vasopressin, L-methionyl-L-tyrosyl-L-phenylalaninamide, has been modified for use in photoaffinity labeling studies of the hormone binding site on neurophysin. The photo-sensitive peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was found by equilibrium dialysis in the absence of light to be reversibly bound to bovine neurophysin II. The dissociation constant, stoichiometry, pH-dependence and competition properties with hormone analogues indicate that the interaction is similar to the interaction of hormone with neurophysin. Covalent complexes resulting from irradiation were fractionated on an affinity matrix. Investigation of the specifically labeled complex which would not bind to the affinity matrix revealed a 1:1 stoichiometry of incorporation and when oxidized, digested with trypsin and subjected to peptide mapping, affinity label remained attached to the peptide mode up of residues 44-66. Details of the site of attachment within this peptide will be studied by sequencing techniques. Irradiation of photoaffinity label with neurophysins from other species produces similar complexes and demonstrates the general applicability of this system.